Structural and functional studies of the interaction of the eukaryotic elongation factor EF-2 with GTP and ribosomes

The structure of the guanosine nucleotide binding site of EF-2 was studied by affinity labelling with the GTP analogue, oxidized GTP (oGTP), and by amino acid sequencing of polypeptides generated after partial degradation with trypsin and N-chlorosuccinimide. Native EF-2 contains two exposed trypsin-sensitive cleavage sites. One site is at Arg66 with a second site at Lys571/Lys572. oGTP was covalently bound to the factor between Arg66 and Lys571. After further cleavage of this fragment with the tryptophan-specific cleavage reagent N-chlorosuccinimide, oGTP was found associated with a polypeptide fragment originating from a cleavage at Trp261 and Trp343. The covalent oGTP . EF-2 complex was capable of forming a high-affinity complex with ribosomes, indicating that oGTP, in this respect, induced a conformation in EF-2 indistinguishable from that produced by GTP. Although GTP could be substituted by non-covalently linked oGTP in the factor and ribosome-dependent GTPase reaction, the factor was unable to utilize the covalently bound oGTP as a substrate. This indicates that the conformational flexibility in EF-2 required for the ribosomal activation of the GTPase was inhibited by the covalent attachment of the nucleotide to the factor. EF-2 cleaved at Arg66 were unable to form the high-affinity complex with ribosomes while retaining the ability to form the low-affinity complex and to hydrolyse GTP. The second cleavage at Lys571/Lys572 was accompanied by a total loss of both the low-affinity binding and the GTPase activity.     

The mechanism of the protein-synthesis elongation cycle in eukaryotes. Effect of ricin on the ribosomal interaction with elongation factors

The functional significance of the post-translocation interaction of eukaryotic ribosomes with EF-2 was studied using the translational inhibitor ricin. Ribosomes treated with ricin showed a decreased rate of elongation accompanied by altered proportions of the different ribosomal phases of the elongation cycle. The content of ribosome-bound EF-2 was diminished by approximately 65% while that of EF-1 was unaffected. The markedly reduced content of EF-2 was caused by an inability of the ricin-treated ribosomes to form high-affinity pre-translocation complexes with EF-2. However, the ribosomes were still able to interact with EF-2 in the form of a low-affinity post-translocation complex. Ricin-treated ribosomes showed an altered ability to stimulate the GTP hydrolysis catalysed by either EF-1 or EF-2. The EF-1-catalysed hydrolysis was reduced by approximately 70%, resulting in a decreased turnover of the quaternary EF-1 X GTP X aminoacyl-tRNA X ribosome complex. In contrast, the EF-2-catalysed hydrolysis was increased by more than 400%, despite the lack of pre-translocation complex formation. The effect was not restricted to empty reconstituted ribosomes since gently salt-washed polysomes also showed an increased rate of GTP hydrolysis. The results indicate that the EF-1- and EF-2-dependent hydrolysis of GTP was activated by a common center on the ribosome that was specifically adapted for promoting the GTP hydrolysis of either EF-1 or EF-2. Furthermore, the results suggest that the GTP hydrolysis catalysed by EF-2 occurred in the low-affinity post-translocation complex.     

Characterization of the ribosomal properties required for formation of a GTPase active complex with the eukaryotic elongation factor 2

The binding stability of the different nucleotide-dependent and -independent interactions between elongation factor 2 (EF-2) and 80S ribosomes, as well as 60S subunits, was studied and correlated to the kinetics of the EF-2- and ribosome-dependent hydrolysis of GTP. Empty reconstituted 80S ribosomes were found to contain two subpopulations of ribosomes, with approximately 80% capable of binding EF-2.GuoPP[CH2]P with high affinity (Kd less than 10(-9) M) and the rest only capable of binding the factor-nucleotide complex with low affinity (Kd = 3.7 x 10(-7) M). The activity of the EF-2- and 80S-ribosome dependent GTPase did not respond linearly to increasing factor concentrations. At low EF-2/ribosome ratios the number of GTP molecules hydrolyzed/factor molecule was considerably lower than at higher ratios. The low response coincided with the formation of the high-affinity complex. At increasing EF-2/ribosome ratios, the ribosomes capable of forming the high-affinity complex was saturated with EF-2, thus allowing formation of the low-affinity ribosome.EF-2 complex. Simultaneously, the GTPase activity/factor molecule increased, indicating that the low-affinity complex was responsible for activating the GTP hydrolysis. The large ribosomal subunits constituted a homogeneous population that interacted with EF-2 in a low-affinity (Kd = 1.3 x 10(-6) M) GTPase active complex, suggesting that the ribosomal domain responsible for activating the GTPase was located on the 60S subunit. Ricin treatment converted the 80S particles to the type of conformation only capable of interacting with EF-2 in a low-affinity complex. The structural alteration was accompanied by a dramatic increase in the EF-2-dependent GTPase activity. Surprisingly, ricin had no effect on the factor-catalyzed GTP hydrolysis in the presence of 60S subunits alone.     

Modification of the accessibility of ribosomal proteins after elongation factor 2 binding to rat liver ribosomes and during translocation

Free- and EF-2-bound 80 S ribosomes, within the high-affinity complex with the non-hydrolysable GTP analog: guanylylmethylenediphosphonate (GuoPP(CH2)P), and the low-affinity complex with GDP, were treated with trypsin under conditions that modified neither their protein synthesis ability nor their sedimentation constant nor the bound EF-2 itself. Proteins extracted from trypsin-digested ribosomes were unambiguously identified using three different two-dimensional gel electrophoresis systems and 5 S RNA release was checked by submitting directly free- and EF-2-bound 80 S ribosomes, incubated with trypsin, to two-dimensional gel electrophoresis. Our results indicate that the binding of (EF-2)-GuoPP[CH2]P to 80 S ribosomes modified the behavior of a cluster of five proteins which were trypsin-resistant within free 80 S ribosomes and trypsin-sensitive within the high-affinity complex (proteins: L3, L10, L13a, L26, L27a). As for the binding of (EF-2)-GDP to 80 S ribosomes, it induced an intermediate conformational change of ribosomes, unshielding only protein L13a and L27a. Quantitative release of free intact 5 S RNA which occurred in the first case but not in the second one, should be related to the trypsinolysis of protein(s) L3 and/or L10 and/or L26. Results were discussed in relation to structural and functional data available on the ribosomal proteins we found to be modified by EF-2 binding.     

Reduced ribosomal binding of eukaryotic elongation factor 2 following ADP-ribosylation. Difference in binding selectivity between polyribosomes and reconstituted monoribosomes

The biological activity of elongation factor 2 (EF-2) following NAD+ - and diphtheria-toxin-dependent ADP-ribosylation was studied (i) in translation experiments using the reticulocyte lysate system and (ii) in ribosomal binding experiments using either reconstituted empty rat liver ribosomes or programmed reticulocyte polysomes. Treatment of the lysates with toxin and NAD+ at a NAD+/ribosome ratio of 4 resulted in a 90% inhibition of the amino acid incorporation rate. The inhibition was overcome by the addition of native EF-2. At this level of inhibition more than 90% of the EF-2 present in the lysates was ADP-ribosylated and the total ribosome association of EF-2 was reduced by approx. 50%. All of the remaining unmodified factor molecules were associated with the ribosomes, whereas only about 3% of the ribosylated factor was ribosome-associated. The nucleotide requirement for the binding of EF-2 to empty reconstituted rat liver ribosomes and programmed reticulocyte polysomes was studied together with the stability of the resulting EF-2 X ribosome complexes using purified 125I-labelled rat liver EF-2. With both types of ribosomes, the complex formation was strictly nucleotide-dependent. Stable, high-affinity complexes were formed in the presence of the non-hydrolysable GTP analogue guanosine 5'-(beta, gamma-methylene)triphosphate (GuoPP[CH2]P). In contrast to the reconstituted ribosomes, GTP stimulated the formation of high-affinity complexes in the presence of polysomes, albeit at a lower efficiency than GuoPP[CH2]P. The formation of high-affinity complexes was restricted to polysomes in the pretranslocation phase of the elongation cycle. Low-affinity post-translocation complexes, demonstrable after fixation, were formed in the presence of GTP, GuoPP[CH2]P and GDP. In polysomes, these complexes involved a different population of particles than did the high-affinity complexes. In the binding experiments using reconstituted or programmed ribosomes, the pretranslocation binding of EF-2 observed in the presence of GuoPP[CH2]P was reduced by approx. 50% after ADP-ribosylation, whereas the post-translocation binding in the presence of GDP was unaltered. The data indicate that the inhibition of translocation caused by diphtheria toxin and NAD+ is mediated through a reduced affinity of the ADP-ribosylated EF-2 for binding to ribosomes in the pretranslocation state.     

The ribosomal stalk binds to translation factors IF2, EF-Tu, EF-G and RF3 via a conserved region of the L12 C-terminal domain

Efficient protein synthesis in bacteria requires initiation factor 2 (IF2), elongation factors Tu (EF-Tu) and G (EF-G), and release factor 3 (RF3), each of which catalyzes a major step of translation in a GTP-dependent fashion. Previous reports have suggested that recruitment of factors to the ribosome and subsequent GTP hydrolysis involve the dimeric protein L12, which forms a flexible "stalk" on the ribosome. Using heteronuclear NMR spectroscopy we demonstrate that L12 binds directly to the factors IF2, EF-Tu, EF-G, and RF3 from Escherichia coli, and map the region of L12 involved in these interactions. Factor-dependent chemical shift changes show that all four factors bind to the same region of the C-terminal domain of L12. This region includes three strictly conserved residues, K70, L80, and E82, and a set of highly conserved residues, including V66, A67, V68 and G79. Upon factor binding, all NMR signals from the C-terminal domain become broadened beyond detection, while those from the N-terminal domain are virtually unaffected, implying that the C-terminal domain binds to the factor, while the N-terminal domain dimer retains its rotational freedom mediated by the flexible hinge between the two domains. Factor-dependent variations in linewidths further reveal that L12 binds to each factor with a dissociation constant in the millimolar range in solution. These results indicate that the L12-factor complexes will be highly populated on the ribosome, because of the high local concentration of ribosome-bound factor with respect to L12.     

Cleavage of a 23S rRNA pseudoknot by phenanthroline-Cu(II)

Studying the intricate folding of rRNA within the ribosome remains a complex problem. Phenanthroline-Cu(II) complexes cleave phosphodiester bonds in rRNA in specific regions, apparently especially where the rRNA structure is constrained in some fashion. We have introduced phenanthroline-copper complexes into 50S Escherichia coli ribosomal subunits and shown specific cleavages in the regions containing nucleotides 60-66 and 87-100. This specificity of cleavage is reduced when the ribosome is heated to 80 degrees C and reduced to background when the ribosomal proteins are extracted and the cleavage repeated on protein-free 23S rRNA. It has been suggested that nucleotides 60-66 and 87-95 in E.coli 23S rRNA are involved in a putative pseudoknot structure, which is supported by covariance data. The paired cleavages of nearly equal intensity of these two regions, when in the ribosome, may further support the existence of a pseudoknot structure in the 100 region of 23S rRNA.     

Effect of ADP-ribosylation and phosphorylation on the interaction of elongation factor 2 with guanylic nucleotides.

Samples of unmodified EF-2, EF-2 ADP-ribosylated with diphtheria toxin and NAD, and/or phosphorylated using ATP and the Ca(2+)-calmodulin dependent kinase III partially purified, were irradiated at 254 nm with 32P-labeled GDP or GTP, and analyzed by one- and two-dimensional gel electrophoresis. By this method we showed that unmodified EF-2 formed a stable complex with GDP but not with GTP, whereas phosphorylated EF-2 and ADP-ribosylated + phosphorylated EF-2 formed stable complexes even in the absence of irradiation, with GTP but not GDP. ADP-ribosylated EF-2 did not form stable complexes with either GDP or GTP. Prior ADP-ribosylation of EF-2 increased its ability to the phosphorylated. These results show that the structures of the two domains containing diphtamide 715 and the phosphorylatable threonines (between Ala 51 and Arg 60) are interdependent; modifications of these residues induce different conformational changes of EF-2 which alter the interactions of the factor with guanylic nucleotides as well with ribosomes.     

The ribosomal binding site for eukaryotic elongation factor EF-2 contains 5 S ribosomal RNA

The possible location of RNA in the ribosomal attachment site for the eukaryotic elongation factor EF-2 was analysed. Stable EF-2 · ribosome complexes formed in the presence of the non-hydrolysable GTP analogue GuoPP[CH₂]P were cross-linked with the short (4 Å between the reactive groups) bifunctional reagent, diepoxybutane. Non-cross-linked EF-2 was removed and the covalent factor-ribosome complex isolated. No interaction between EF-2 and 18 S or 28 S rRNA could be demonstrated. However, density gradient centrifugation of the cross-linked ribosomal complexes showed an increased density (1.25 g/cm³) of the factor, as expected from a covalent complex between EF-2 and a low-molecular-weight RNA species. Treatment of the covalent ribosome-factor complexes with EDTA released approx 50% of the cross-linked EF-2 from the ribosome together with the 5 S rRNA · protein L5 complex. Furthermore, the complex co-migrated with the 5S rRNA · L5 particle in sucrose gradients. Polyacrylamide gel electrophoresis showed that EF-2 was directly linked to 5 S rRNA in the 5 S rRNA · L5 complex, as well as in the complexes isolated by density gradient centrifugation. No traces of 5.8 S rRNA or tRNA could be demonstrated. The data indicate that the ribosomal binding domain for EF-2 contains the 5 S rRNA · protein L5 particle and that EF-2 is located in close proximity to 5 S rRNA within the EF-2 · GuoPP[CH₂]P · ribosome complex.     

Identification of the major Mr 100,000 substrate for calmodulin-dependent protein kinase III in mammalian cells as elongation factor-2

The major substrate for Ca2+/calmodulin-dependent protein kinase III in mammalian cells is a species of Mr 100,000 that has a primarily cytoplasmic localization. This substrate has now been identified as elongation factor-2 (EF-2), a protein that catalyzes the translocation of peptidyl-tRNA on the ribosome. The amino acid sequence of 18 residues from the N-terminal of the Mr 100,000 CaM-dependent protein kinase III substrate purified from rat pancreas was found to be identical to the N-terminal sequence of authentic rat EF-2 as previously deduced from nucleic acid sequencing of a cDNA (Kohno, K., Uchida, T., Ohkubo, H., Nakanishi, S., Nakanishi, T., Fukui, T., Ohtsuka, E., Ikehara, M., and Okada, Y. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 4978-4982). CaM-dependent protein kinase III phosphorylated EF-2 in vitro with a stoichiometry of approximately 1 mol/mol on a threonine residue. Amino acid sequencing of the purified tryptic phosphopeptide revealed that this threonine residue lies within the sequence: Ala-Gly-Glu-Thr-Arg-Phe-Thr-Asp-Thr-Arg (residues 51-60 of EF-2). The Mr 100,000 protein was stoichiometrically ADP-ribosylated in vitro by the addition of diphtheria toxin and NAD. The Mr 100,000 protein was photoaffinity labeled with a GTP analog and the protein had an endogenous GTPase activity that could be stimulated by the addition of salt-washed ribosomes. These properties are all characteristic of EF-2. Dephospho-EF-2 could support poly(U)-directed polyphenylalanine synthesis in a reconstituted elongation system when combined with EF-1. In the same system, phospho-EF-2 was virtually inactive in supporting polypeptide synthesis; this effect could be reversed by dephosphorylation of phospho-EF-2. These results suggest that intracellular Ca2+ inhibits protein synthesis in mammalian cells via CaM-dependent protein kinase III-catalyzed phosphorylation of EF-2.     

Eukaryotic protein elongation factors

In eukaryotes, peptide chain elongation is mediated by elongation factors EF-1 and EF-2. EF-1 is composed of a nucleotide-binding protein EF-1 alpha, and a nucleotide exchange protein complex, EF-1 beta gamma, while EF-2 catalyses the translocation of peptidyl-tRNA on the ribosome. Elongation factors are highly conserved among different species and may be involved in functions other than protein synthesis, such as organization of the mitotic apparatus, signal transduction, developmental regulation, ageing and transformation. Yeast contains a third factor, EF-3, whose structure and function is not yet well understood.     

The solution structure of the guanine nucleotide exchange domain of human elongation factor 1beta reveals a striking resemblance to that of EF-Ts from Escherichia coli.

BACKGROUND: In eukaryotic protein synthesis, the multi-subunit elongation factor 1 (EF-1) plays an important role in ensuring the fidelity and regulating the rate of translation. EF-1alpha, which transports the aminoacyl tRNA to the ribosome, is a member of the G-protein superfamily. EF-1beta regulates the activity of EF-1alpha by catalyzing the exchange of GDP for GTP and thereby regenerating the active form of EF-1alpha. The structure of the bacterial analog of EF-1alpha, EF-Tu has been solved in complex with its GDP exchange factor, EF-Ts. These structures indicate a mechanism for GDP-GTP exchange in prokaryotes. Although there is good sequence conservation between EF-1alpha and EF-Tu, there is essentially no sequence similarity between EF-1beta and EF-Ts. We wished to explore whether the prokaryotic exchange mechanism could shed any light on the mechanism of eukaryotic translation elongation. RESULTS: Here, we report the structure of the guanine-nucleotide exchange factor (GEF) domain of human EF-1beta (hEF-1beta, residues 135-224); hEF-1beta[135-224], determined by nuclear magnetic resonance spectroscopy. Sequence conservation analysis of the GEF domains of EF-1 subunits beta and delta from widely divergent organisms indicates that the most highly conserved residues are in two loop regions. Intriguingly, hEF-1beta[135-224] shares structural homology with the GEF domain of EF-Ts despite their different primary sequences. CONCLUSIONS: On the basis of both the structural homology between EF-Ts and hEF-1beta[135-224] and the sequence conservation analysis, we propose that the mechanism of guanine-nucleotide exchange in protein synthesis has been conserved in prokaryotes and eukaryotes. In particular, Tyr181 of hEF-1beta[135-224] appears to be analogous to Phe81 of Escherichia coli EF-Ts.     

Sites of the NUDT9-H domain critical for ADP-ribose activation of the cation channel TRPM2

TRPM2 is a cation channel unique within the transient receptor potential family because of its gating by ADP-ribose (ADPR). ADPR gating is enabled by a cytosolic C-terminal Nudix box sequence motif embedded into a region homologous to the NUDT9 ADPR pyrophosphatase. A recently discovered splice variant of TRPM2 (TRPM2-DeltaC) lacks 34 amino acid residues in the NUDT9 domain and is insensitive to ADPR. To analyze in detail which parts of the deleted sequence (DeltaC-stretch) are critical for ADPR gating, we tested mutants that lacked 19, 25, and 29 amino acid residues in the N-terminal part or had amino acid residues substituted in the remaining C-terminal part of the DeltaC-stretch. All of these mutants displayed typical ADPR-induced currents. However, the deletion or substitution of the amino acid residue Asn-1326 immediately downstream of the DeltaC-stretch abrogated ADPR gating. We furthermore analyzed the mutation I1405E/L1406F in the Nudix box of TRPM2, because a considerably decreased AD-PRase activity of the TRPM2 NUDT9-H protein in comparison to the NUDT9 pyrophosphatase has been attributed to the reverse exchange EF --> IL. The I1405E/L1406F variant of TRPM2 failed to respond to ADPR even at concentrations up to 10 mM. We concluded that the DeltaC-stretch contains no individual amino acid residues essential for ADPR gating but may act as a spacer segment stabilizing a conformation necessary for the essential residue Asn-1326 to interact with other channel regions. Enhancing the enzymatic activity of the Nudix box abolishes the ADPR gating of TRPM2, pointing to the requirement of prolonged binding rather than degradation.     

Expression of elongation factor 1 beta' in Escherichia coli and its interaction with elongation factor 1 alpha from silk gland.

Silk gland elongation factor 1 (EF-1) consists of four subunits: alpha, beta, beta', and gamma. EF-1 beta beta' gamma catalyzes the exchange of GDP for GTP on EF-1 alpha and stimulates the binding of EF-1 alpha-dependent aminoacyl-tRNA to ribosomes. The carboxy-terminal regions of the EF-1 beta subunits from various species are highly conserved. We examined the region of EF-1 beta' that binds to EF-1 alpha by in vitro binding assays, and examined the GDP/GTP exchange activity using deletion mutants of a GST-EF1 beta' fusion protein. We thereby suggested a pivotal amino acid region, residues 189-222, of EF-1 beta' for binding to EF-1 alpha.     

Mutant proteins of human interleukin 2. Renaturation yield, proliferative activity and receptor binding

Muteins, i.e. proteins altered by mutation of their genes, of interleukin 2 (Il2) were generated by oligonucleotide-directed mutagenesis in vitro. All acidic and basic residues conserved between man and mouse were exchanged as well as four lipophilic residues contained within four hydrophobic segments of the protein. The muteins were produced in Escherichia coli and submitted to a renaturation and purification protocol, before bioactivity and receptor binding of each of them was determined. All muteins besides two (K44/T125 and Q110/T125) could be renatured and purified. One mutein (K94/T125) exhibited a more than tenfold-improved renaturation yield. One amino exchange (Asp-20 to Asn) resulted in an about 20-fold reduction in proliferative activity and high-affinity receptor binding. The binding to the low-affinity Il2-binding protein (Tac antigen) was unimpaired. A second exchange (Arg-38 to Gln) had no effect on proliferative activity. The binding to both the high- and the low-affinity receptor, however, was reduced about 20-fold. Preliminary trials on the stability of these muteins by guanidinium hydrochloride denaturation studies detected no differences between wild-type interleukin 2 and muteins. It is suggested that Asp-20 forms part of the binding site for the large receptor subunit whereas Arg-38 is involved in the contact site to the small subunit.     

Structure-function analysis of the reactive site in the first Kunitz-type domain of human tissue factor pathway inhibitor-2

Human tissue factor pathway inhibitor-2 (TFPI-2) is a Kunitz-type proteinase inhibitor that regulates a variety of serine proteinases involved in coagulation and fibrinolysis through their non-productive interaction with a P(1) residue (Arg-24) in its first Kunitz-type domain (KD1). Previous kinetic studies revealed that TFPI-2 was a more effective inhibitor of plasmin than several other serine proteinases, but the molecular basis for this specificity was unclear. In this study, we employed molecular modeling and mutagenesis strategies to produce several variants of human TFPI-2 KD1 in an effort to identify interactive site residues other than the P(1) Arg that contribute significantly to its inhibitory activity and specificity. Molecular modeling of KD1 based on the crystal structure of bovine pancreatic trypsin inhibitor revealed that KD1 formed a more energetically favorable complex with plasmin versus trypsin and/or the factor VIIa-tissue factor complex primarily due to strong ionic interactions between Asp-19 (P(6)) and Arg residues in plasmin (Arg-644, Arg-719, and Arg-767), Arg-24 (P(1)) with Asp-735 in plasmin, and Arg-29 (P(5)') with Glu-606 in plasmin. In addition, Leu-26 through Leu-28 (P(2)'-P(4)') in KD1 formed strong van der Waals contact with a hydrophobic cluster in plasmin (Phe-583, Met-585, and Phe-587). Mutagenesis of Asp-19, Tyr-20, Arg-24, Arg-29, and Leu-26 in KD1 resulted in substantial reductions in plasmin inhibitory activity relative to wild-type KD1, but the Asp-19 and Tyr-20 mutations revealed the importance of these residues in the specific inhibition of plasmin. In addition to the reactive site residues in the P(6)-P(5)' region of KD1, mutation of a highly conserved Phe at the P(18)' position revealed the importance of this residue in the inhibition of serine proteinases by KD1. Thus, together with the P(1) residue, the nature of other residues flanking the P(1) residue, particularly at P(6) and P(5)', strongly influences the inhibitory activity and specificity of human TFPI-2.     

Identification of the sites in the eukaryotic elongation factor 1 alpha involved in the binding of elongation factor 1 beta and aminoacyl-tRNA.

In this article we report the identification of the sites which are involved in the binding of the GDP-exchange factor EF-1 beta and aminoacyl tRNA to the alpha-subunit of the eukaryotic elongation factor 1 (EF-1) from Artemia. For this purpose the polypeptide chain of EF-1 alpha, having 461 amino acid residues, was proteolytically cleaved into large fragments by distinct proteases. Under well defined conditions, a mixture of two large fragments, free from intact EF-1 alpha and with molecular masses of 37 kDa and 43 kDa, was obtained. The 37-kDa and 43-kDa fragments comprise the residues 129-461 and 69-461, respectively. However, in aqueous solution and under non-denaturing conditions, the mixture still contained a short amino-terminal peptide, encompassing the residues 1-36, that remained tightly bound. The ability of the mixture of the 37+43-kDa fragments, including this amino-terminal peptide 1-36, to bind GDP or to facilitate aminoacyl tRNA binding to salt-washed ribosomes was severely reduced, compared to intact EF-1 alpha. However, both of these complexes were able to bind to the GDP-exchange-stimulating subunit EF-1 beta. A 30-kDa fragment, comprising the residues 1-287, was generated after treatment of the protein with endoproteinase Glu-C. This fragment contained the complete guanine nucleotide binding pocket. Although it was able to bind GDP and to transport aminoacyl tRNA to the ribosome, no affinity towards EF-1 beta was observed. We propose that the guanine-nucleotide-exchange stimulation by EF-1 beta is induced through binding of this factor to the carboxy-terminal part of EF-1 alpha. As a result, a decreased susceptibility towards trypsin of the guanine-nucleotide-binding pocket of EF-1 alpha, especially in the region of its presumed effector loop is induced.     

Structural basis for different substrate specificities of two ADP-ribose pyrophosphatases from Thermus thermophilus HB8

ADP-ribose (ADPR) is one of the main substrates of Nudix proteins. Among the eight Nudix proteins of Thermus thermophilus HB8, we previously determined the crystal structure of Ndx4, an ADPR pyrophosphatase (ADPRase). In this study we show that Ndx2 of T. thermophilus also preferentially hydrolyzes ADPR and flavin adenine dinucleotide and have determined its crystal structure. We have determined the structures of Ndx2 alone and in complex with Mg2+, with Mg2+ and AMP, and with Mg2+ and a nonhydrolyzable ADPR analogue. Although Ndx2 recognizes the AMP moiety in a manner similar to those for other ADPRases, it recognizes the terminal ribose in a distinct manner. The residues responsible for the recognition of the substrate in Ndx2 are not conserved among ADPRases. This may reflect the diversity in substrate specificity among ADPRases. Based on these results, we propose the classification of ADPRases into two types: ADPRase-I enzymes, which exhibit high specificity for ADPR; and ADPRase-II enzymes, which exhibit low specificity for ADPR. In the active site of the ternary complexes, three Mg2+ ions are coordinated to the side chains of conserved glutamate residues and water molecules. Substitution of Glu90 and Glu94 with glutamine suggests that these residues are essential for catalysis. These results suggest that ADPRase-I and ADPRase-II enzymes have nearly identical catalytic mechanisms but different mechanisms of substrate recognition.     

Replacement of L7/L12.L10 protein complex in Escherichia coli ribosomes with the eukaryotic counterpart changes the specificity of elongation factor binding.

The L8 protein complex consisting of L7/L12 and L10 in Escherichia coli ribosomes is assembled on the conserved region of 23 S rRNA termed the GTPase-associated domain. We replaced the L8 complex in E. coli 50 S subunits with the rat counterpart P protein complex consisting of P1, P2, and P0. The L8 complex was removed from the ribosome with 50% ethanol, 10 mM MgCl(2), 0.5 M NH(4)Cl, at 30 degrees C, and the rat P complex bound to the core particle. Binding of the P complex to the core was prevented by addition of RNA fragment covering the GTPase-associated domain of E. coli 23 S rRNA to which rat P complex bound strongly, suggesting a direct role of the RNA domain in this incorporation. The resultant hybrid ribosomes showed eukaryotic translocase elongation factor (EF)-2-dependent, but not prokaryotic EF-G-dependent, GTPase activity comparable with rat 80 S ribosomes. The EF-2-dependent activity was dependent upon the P complex binding and was inhibited by the antibiotic thiostrepton, a ligand for a portion of the GTPase-associated domain of prokaryotic ribosomes. This hybrid system clearly shows significance of binding of the P complex to the GTPase-associated RNA domain for interaction of EF-2 with the ribosome. The results also suggest that E. coli 23 S rRNA participates in the eukaryotic translocase-dependent GTPase activity in the hybrid system.     

Effects of phosphorylation by CAK on cyclin binding by CDC2 and CDK2.

The cyclin-dependent protein kinases (CDKs) are activated by association with cyclins and by phosphorylation at a conserved threonine residue by the CDK-activating kinase (CAK). We have studied the binding of various human CDK and cyclin subunits in vitro, using purified proteins derived from baculovirus-infected insect cells. We find that most CDK-cyclin complexes known to exist in human cells (CDC2-cyclin B, CDK2-cyclin A, and CDK2-cyclin E) form with high affinity in the absence of phosphorylation or other cellular components. One complex (CDC2-cyclin A) forms with high affinity only after CAK-mediated phosphorylation of CDC2 at the activating threonine residue. CDC2 does not bind with high affinity to cyclin E in vitro, even after phosphorylation of the CDC2 subunit. Thus, phosphorylation is of varying importance in the formation of high-affinity CDK-cyclin complexes.